Mycobacterium tuberculosis (Mtb) is an intracellular pathogen. Paradoxically, macrophages—whose purpose is to destroy such pathogens—provide both a primary niche for the bacterium's survival and replication, as well as acting as the major mediator of host protection through T cell-mediated activation of uninfected macrophages. Following infection, Mtb are phagocytosed by alveolar macrophages in the lung and subsequently colonize the underlying epithelial layer, which triggers recruitment of mononuclear cells from neighboring blood vessels that serve as host cells for multiplying bacteria and causing a condition of Tuberculosis (TB).
TB is a common and often lethal infectious disease caused by the Mycobacterium genus of bacteria, typically Mtb. According to the World Health Organization, one-third of the world population may be infected with Mtb and each year, is it estimated that over 8 million new cases, and almost 1.5 million deaths are caused by TB. This disease is particularly common in low to middle-income countries, accounting for roughly 80% of reported disease cases. Furthermore, the high prevalence of HIV in areas such as sub-Saharan Africa greatly adds to TB's lethality.
TB is air transmissible and, therefore, easily spreads between individuals through respiratory fluid droplets. Most Mtb infections are asymptomatic, latent TB infections (LTBI), however, some of these infections can eventually progress to active TB infections. TB typically infects the pulmonary system of an afflicted individual; however, the disease can also spread through the body causing extrapulmonary TB (EPTB). Roughly one in ten latent TB infections progresses to an active disease, which, if left untreated, typically kills more than 50% of those infected.
Given TB's high prevalence and associated deaths, fast diagnosis and treatment of active TB is of paramount importance. However, present diagnostic assays are often inaccurate, and are unable to distinguish between persons in the latent stage of the disease and those in the active stage. Moreover, available diagnostic tests are many times unable to distinguish between individuals who have been immunized, and individuals infected with TB.
Currently, the two most common methods of detecting Mtb infection are the sputum acid-fast bacilli smear microscopy test (AFB) and the tuberculin skin test (TST). In AFB smears, sputum is collected from patients and examined microscopically after a bacterial staining procedure. Although AFB can produce presumptive results in as little as a few hours, the test suffers from poor sensitivity. AFB also fails to identify TB patients having little to no Mtb in their sputum or those patients who are unable to produce sputum. This is especially common in young children or HIV infected patients. Further, Mtb replicates slowly, making positive identification of Mtb in cultures lengthy, ranging from days to weeks.
TST is a composite measure of cell-mediated immunity in response to TB antigen (PPD) stimulation, which is injected under the skin of a patient. However, it may take 2 to 3 days before the results can be obtained and frequently delivers false positive or false negative results. Also, this test does not distinguish latent infection from active disease, which is important in a diagnostic setting. Additionally, interferon-gamma based tests have also been developed. While these tests can provide rapid detection of Mtb, they too suffer from lack of sensitivity and specificity in certain situations. Moreover, both the cost of such tests, as well as the required expertise to perform such tests can be prohibitive.
Therefore, a need exists for a diagnostic and prognostic assay to identify those at risk for TB and capable of transmitting Mtb to other individuals. The detection of Mtb biomarkers in a patient's bodily fluid (blood, urine, etc.) provides such an opportunity. Therefore, the identification of specific and easily measured extracellular vesicle Mtb biomarkers may have a significant impact on global Mtb diagnosis and treatment. The present disclosure addresses these needs.